Use of Microfluidics Technology to Model Pathogenic Protein Seeding in Neurodegeneration in vitro Dr Eve Corrie,1 Dr Rebecca Kelly,1 Dr Matthieu Trigano,1 and Dr Emma V. Jones1 1Medicines Discovery Catapult, Block 35, Alderley Park, Cheshire, SK10 4ZF, UK.
Introduction Many neurodegenerative diseases are associated with the presence of misfolded, aggregating proteins within the brain, leading to cytotoxicity and cell death. The prion hypothesis states that these toxic species spread through the brain via anatomically connected regions, leading to widespread neurodegeneration. Examples of prion-like proteins in neurodegeneration: Native protein • α-synuclein – Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy • β-amyloid – Alzheimer’s disease • TDP43 – Amyotrophic lateral sclerosis, frontotemporal dementia • Tau – Alzheimer’s disease, corticobasal degeneration, frontotemporal dementia, chronic traumatic encephalopathy, progressive supranuclear palsy
Pathogenic misfolded protein
Native protein
α-synuclein Lewy Body1
Protein aggregate
Compartmentalised Microfluidics Devices for Neuronal Culture •
“Donor” “Acceptor”
• • • eNuvio Omega4 device
Two open chambers connected by 10µm microchannels – narrow enough to maintain separation of cell bodies but allow projection of neurites between chambers Fluidic isolation maintained by asymmetric volume loading to ensure unidirectional flow of fluid Neuron → neuron Neuron → non-neuronal cell (e.g. glia, peripheral cells, myocytes)
Projection of iPSC-derived glutamatergic cortical neurons through microchannels, imaged at 2 weeks in culture • ioGlutamatergic neurons (bit.bio) were labelled with live neuron dye Neurofluor NeuO • Axons can be visualised within the channels and projecting well into the adjacent chamber
Inter-neuronal Seeding of Fluorescent α-synuclein Preformed Fibrils
Donor chamber – PFFs directly applied Neurofluor NeuO α-synuclein PFFs
Neurofluor NeuO
α-synuclein PFFs
0s
Live imaging showing internalisation and fast axonal transport of Alexa Fluor 594-labelled α-synuclein PFFs (a single puncta over time marked by arrow) applied to one “donor” chamber of microfluidics device • •
‘3.25s
• •
‘6.5s
• ‘9.75s
•
Acceptor chamber – seeded PFFs
24h
iPSC-derived glutamatergic cortical neurons were plated in both chambers of a microfluidics device Fluorescently labelled α-synuclein preformed fibrils (PFFs) were added to a fluidically isolated “donor” chamber of the microfluidics device Neurons were labelled with Neurofluor NeuO and imaged live over time following PFF addition Uptake of PFFs could be detected within neurons after direct application to the donor chamber Fast axonal transport of PFF positive puncta could be visualised within the microchannels of the microfluidics device at 24 hours after PFF addition Progressive accumulation of PFFs was then detected in neurons in the connecting “acceptor” compartment over time
48h
Live imaging showing progressive accumulation of Alexa Fluor 594- 72h labelled α-synuclein PFFs in cell bodies (PFF-positive neurons shown by arrows) of the “acceptor” chamber of microfluidics device over time 2 weeks after PFF addition
Phosphorylation and Aggregation of Native α-synuclein Induced by Exposure to Seeded Preformed Fibrils
4
2.3 2
0
- PFF
12
9.8
10 8 6 4 2
0
Conclusions
Compartmentalised cocultures of iPSC-derived glutamatergic cortical neurons and iPSC-derived microglia can be used to assess the effects of seeded α-synuclein PFFs on neuroinflammation
•
α-synuclein PFFs
Iba1 β-tubulin
Integrated intensity of fluorescent α-synuclein PFFs
PFF uptake after direct addition to iPSC-microglia
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3×10 6
0µg/ml 5µg/ml 10µg/ml
2×10 6
• 1×10 6
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20
40
60
80
Time (hours)
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However, when α-synuclein PFFs were added to a neuronal “donor” chamber with microglia in the “acceptor” chamber, no seeded PFFs could be detected in microglia The small amounts of α-synuclein trafficked by axonal transport may be degraded by the microglia, preventing accumulation and detection by confocal imaging Cytokines released into the media in response to seeded α-synuclein will be assessed to determine microglial activation state
1 Carol F. Lippa, Marie L. Schmidt, Virginia M. Lee, & John Q. Trojanowski (1999) ‘Dementia with Lewy bodies’, Neurology, 52(4), pp. 893–893.
0.8 Acceptor chamber
0
Donor chamber
0
Neuron/Microglia Coculture in Microfluidics Devices ioMicroglia (bit.bio) were shown to internalise fluorescently labelled α-synuclein PFFs, as shown by confocal and Incucyte imaging
+ PFF
3 weeks after PFF addition
- PFF
•
0 Acceptor chamber
2 weeks
0
Donor chamber
0
At 2 weeks, phosphorylated αsynuclein was detected in the “donor” chamber neurons only At 3 weeks, phosphorylated α- 3 weeks synuclein was additionally detected in neurons of the “acceptor” chamber
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6
Acceptor chamber
•
Phospho-S129 α-synuclein β-tubulin
8
Acceptor chamber
Phospho-S129 α-synuclein β-tubulin
Acceptor chamber – seeded PFFs
10
Donor chamber
Lewy body-like perinuclear accumulation of phosphorylated serine 129 α-synuclein (arrows) in response to directly applied and seeded PFFs
Donor chamber – PFFs directly applied
% pS129 positive
Microfluidics devices were fixed at 2 or 3 weeks after PFF addition to “donor” chamber Immunocytochemistry for β-tubulin and phosphorylated serine 129 α-synuclein was carried out
% pS129 positive
• •
12
Donor chamber
‘13s
+ PFF
Uptake, axonal transport, seeding and subsequent phosphorylation and aggregation of α-synuclein can be modelled in microfluidics devices The effect of seeded α-synuclein to other cell types such as microglia could be assayed This technique could be widely applied to model the seeding of other aggregating neurodegenerative proteins